The ubiquitin pathway is a key regulatory system in eukaryotes. Ubiquitin is presently the only known post-translational marker for protein degradation in nature. Here we describe a new proteolytic pathway that employs a ubiquitin-like protein termed Rub1. Using a genetic selection, we have identified an engineered protein, JL1, whose degradation in yeast is strictly dependent on RUB1. JL1 contains a Ura3 reporter protein linked to a nine-residue extension which appears to constitute a RUB1-dependent degradation signal. Using the Ura phenotypes of JL1, the activity of the Rub1 pathway can be followed by simple plate assays, allowing us to dissect the pathway genetically. Although Rub1 forms covalent conjugates in vivo, ubiquitin-activating enzyme fails to recognize Rub1. These data suggest that the Rub1 conjugation pathway is distinct from that of ubiquitin. Ubc13, which was previously thought to be involved in ubiquitination, is apparently the conjugating enzyme for Rub1. In contrast, the proteasome, a post- conjugative component of the ubiquitin pathway, is required for the degradation of Rub1-conjugated proteins. Thus, in eukaryotes, there exist two pathways for targeted protein degradation, which converge at the proteasome where their substrates are degraded. Rub1 has close homologues in plants and mammals, suggesting that the Rub1 proteolytic pathway is general to the eukaryotic kingdom. The discovery of the Rub1 degradation pathway and the demonstration that the proteasome recognizes and degrades substrates tagged with a protein other than ubiquitin has significant implications for our view of eukaryotic protein degradation. This proposal is focused on identification of the components of the Rub1 pathway, which will be facilitated by the existence of the powerful JL1- based genetic assay for pathway function. Specific Aims are as follows: Aim 1. Genetic identification of Rub1-conjugating factors and in vitro reconstitution of Rub1-JL1 conjugation from purified, cloned components. Aim 2. Mutational dissection of the functional elements of the Rub1 protein. Aim 3. Genetic identification of the Rub1 receptor. Aim 4. Biochemical confirmation of the identity of the Rub1 receptor.